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D Naville

Publications and source records attributed to D Naville.

At least 37 records · Page 2Linked to original sources
PubMed1996

[Mutations of ACTH receptor gene and familial syndrome of glucocorticoid deficiency].

Familial isolated glucocorticoid deficiency syndrome is characterized by low cortisol plasma levels despite high ACTH levels without any stimulation of steroid production after ACTH administration. However, the mineralocorticoid function is well-preserved in this syndrome which indicates a specific resistance to ACTH. Recent cloning of the ACTH receptor allowed to study this receptor in this particular syndrome. After studying sixteen affected families, we have found three mutations in two patients from non-related families. One of these patients was a double heterozygote compound (C251F, G217fs) while the other one was homozygote for another mutation D107N. The mutant receptors were expressed in vitro in transfected M3 cells (S91 Cloudman cells) which represents a working expression system to express the ACTH receptor. Production of intracellular cyclic AMP was calculated in the presence of increasing concentrations of ACTH. The EC50 values were estimated (C251F: 3.5 +/- 0.9 x 10(-9) M, D107N: 3.0 +/- 0.9 x 10(-9) M, G217fs: 4.8 +/- 0.9 x 10(-9) M) and comparison with the value obtained for the wild type ACTH receptor (5.1 +/- 0.9 x 10(-10) M) indicates a clear 6 to 9 shift to the right due to an impaired function of these mutant receptors. Such results were expected for the G217fs mutation, and could be explained by a decrease in ligand affinity or an impaired coupling to adenylate cyclase in the case of amino acid substitutions. A total of twelve mutations has been described in the literature although eight of them have not been tested in vitro until now.

Gene Expression↗
PubMed1994

Characterization of the transcription start site of the ACTH receptor gene: presence of an intronic sequence in the 5'-flanking region.

Corticotropin (ACTH) regulates glucocorticoid production through specific receptors on the adrenal cortex. Analysis of the ACTH receptor mRNA in human adrenal has revealed the presence of five transcripts ranging from 1.8 to 11 kilobases (kb). Characterization of the 5'-untranslated regions (UTRs) of the ACTH receptor mRNA demonstrated the presence of one major initiation site of transcription 177 bp away from the ATG codon. Analysis of this 5' sequence showed a perfect alignment with the previously described genomic sequence until position -128 bp from the ATG. The upstream 49-bp sequence was divergent, suggesting the occurrence of a splicing and indicating the presence of an intronic sequence in the UTRs, as well as the presence of an upstream exon containing this 49-bp sequence and located at least 1.8 kb away from the exon encoding the protein.

Adrenal Glands↗
PubMed1994

Regulation of corticotropin and steroidogenic enzyme mRNAs in human fetal adrenal cells by corticotropin, angiotensin-II and transforming growth factor beta 1.

Using cultured human fetal adrenal cells, we have investigated the basal secretion of cortisol and dehydroepiandrosterone sulfate (DHAS) and the effect of corticotropin (ACTH), angiotensin-II (A-II) and transforming growth factor beta 1 (TGF beta 1) on the secretion of these steroids and on the mRNA levels of ACTH receptor (ACTHR), cytochrome P-450scc (cholesterol side-chain cleavage), P450 17 alpha (17 alpha-hydroxylase/17-20 lyase) and 3 beta-HSD (3 beta-hydroxysteroid dehydrogenase). The basal DHAS/cortisol ratio declined progressively between 12.5 and 21 weeks. ACTH treatment enhanced the secretion of cortisol and to a lesser extent that of DHAS, and increased the steroidogenic response to an acute stimulation with ACTH. These changes were associated with increased mRNA levels of ACTHR and of the steroidogenic enzymes. A-II treatment also increased the secretion of both DHAS and cortisol, but less than ACTH, enhanced the responsiveness to ACTH and increased ACTHR, P450scc and P450 17 alpha mRNA levels. In contrast, TGF beta 1 alone or together with ACTH decreased DHAS secretion, but not cortisol secretion. Moreover, TGF beta 1 had no effect on ACTHR and P450scc mRNA levels, decreased by about 50% the mRNA levels of P450 17 alpha both in the absence or presence of ACTH, but enhanced the stimulatory effects of ACTH on 3 beta-HSD mRNA. These results, along with those previously reported, suggest that both A-II and TGF beta may play a role in fetal adrenal function. In addition, they show that the effects of both peptides are qualitatively different from, even sometimes opposite to, those previously reported in bovine and ovine adrenal cells.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Regulation of corticotropin receptor number and messenger RNA in cultured human adrenocortical cells by corticotropin and angiotensin II.

The regulation of ACTH receptor binding sites and mRNA by ACTH and angiotensin II (A-II) was studied using cultured human adrenal fasciculata reticularis cells (HAC). These cells expressed two major ACTH receptor transcripts of 1.8 and 3.4 kb and three minor ones of 4, 7, and 11 kb. ACTH increased the levels of all these transcripts in a time- and dose-dependent manner. At a maximal concentration of 10(-8) M, ACTH enhanced 21- and 4-fold the level of ACTH receptor mRNA and the number of receptors per cell, respectively. Pretreatment of HAC with A-II produced a dose-dependent enhancement of ACTH receptor mRNA that was associated with an increase of both ACTH receptor number and responsiveness to this hormone. The effects of A-II were completely blocked by an AT1 receptor subtype antagonist but not by an AT2 antagonist. The effects of ACTH together with A-II on ACTH receptor mRNA were greater than those induced by each hormone alone. These results show that ACTH receptor number and mRNA are positively regulated by the two main hormones (ACTH and A-II) which, in vivo, regulate adrenocortical functions. In addition, they also show that HAC are a target for A-II. Thus, regulation of ACTH receptors may be one mechanism by which ACTH and A-II regulate adrenocortical functions under both normal and pathological conditions.

Adrenal Cortex↗
PubMed1994

Effects of transforming growth factor-beta 1 on human adrenocortical fasciculata-reticularis cell differentiated functions.

Transforming growth factor-beta 1 (TGF beta 1) has been reported to have a strong inhibitory effect on the specific function of adrenal cells of several species. In the present study, we examined the effects of TGF beta 1 on cultured human fasciculata-reticularis cells. TGF beta 1 alone had no effect on ACTH receptor messenger ribonucleic acid (mRNA) levels and was unable to reduce the strong stimulatory effects of ACTH on its own receptor. However, TGF beta 1 enhanced angiotensin-II type 1 receptor mRNA and binding sites. Treatment with TGF beta 1 increased significantly the levels of 3 beta-hydroxysteroid dehydrogenase mRNA, reduced those of cytochrome P-450 17 alpha-hydroxylase mRNA, and had no effect on cholesterol side-chain cleavage cytochrome P-450 mRNA. Whatever the experimental condition, TGF beta 1 did not reduce basal or ACTH-stimulated cortisol production, but the production of dehydroepiandrosterone sulfate of TGF beta 1-treated cells was always decreased. The effects of TGF beta 1 on 3 beta-hydroxysteroid dehydrogenase mRNA and dehydroepiandrosterone sulfate were opposite the change observed at the time of adrenarche. As adrenal cells express TGF beta 1 mRNA, it is tempting to postulate that a local diminution of TGF beta 1 might be involved in adrenarche. Our findings also illustrate the specific species differences and, therefore, the caution to extrapolate to humans the results observed in other species.

Adrenal Cortex↗
PubMed1993

Characterization and regulation of the angiotensin II type-1 receptor (binding and mRNA) in human adrenal fasciculata-reticularis cells.

The classical concept of human adrenal physiology indicates that only glomerulosa cells are the target of A-II. Herein, we demonstrated that cultured human adrenal fasciculata-reticularis cells were also responsive to this hormone. Indeed, these cells contained high affinity (Kd = 0.9-1.1 nM) and low capacity (8,000-13,000 sites/cell) A-II receptors, and more than 95% of them were of the type-1. These AT1 receptors are functional since A-II was able to increase cortisol production after 48 h of treatment. These effects were inhibited by losartan, an AT1 antagonist, but not by CGP42112A, an AT2 antagonist. The expression of the type-1 A-II receptor mRNA was detected in the whole adrenal in both adult and fetus, and in cultured human adrenal fasciculata-reticularis cells. In these cells A-II negatively regulated AT1 receptor mRNA, and this effect was also mediated through the AT1 receptor subtype.

Adult↗
PubMed1993

Regulation of expression of the 3 beta-hydroxysteroid dehydrogenases of human placenta and fetal adrenal.

The appropriate expression of 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase (3 beta-HSD) is vital for mammalian reproduction, fetal growth and life maintenance. Several isoforms of 3 beta-HSD, the products of separate genes, have been identified in various species including man. Current investigations are targeted toward defining the processes that regulate the levels of specific isoforms in various steroidogenic tissues of man. High levels of expression of 3 beta-HSD were observed in placental tissues. It has been generally considered that the multinucleated syncytiotrophoblastic cells are the principal sites of 3 beta-HSD expression and, moreover, that 3 beta-HSD expression is intimately associated with cyclic AMP-promoted formation of syncytia. Herein we report the presence of 3 beta-HSD immunoreactive and mRNA species in uninucleate cytotrophoblasts in the chorion laeve, similar to that in syncytia but not cytotrophoblast placenta. In vitro, 3 beta-HSD levels in chorion laeve cytotrophoblasts were not increased with time nor after treatment with adenylate cyclase activators, whereas villous cytotrophoblasts spontaneously demonstrated progressive, increased 3 beta-HSD expression. Moreover, 3 beta-HSD synthesis appeared to precede morphologic syncytial formation. Thus high steroidogenic enzyme expression in placenta is not necessarily closely linked to formation of syncytia. Both Western immunoblot and enzymic activity analyses also indicated that the 3 beta-HSD expressed in these cytotrophoblastic populations was the 3 beta-HSD type I gene product (M(r), 45K) and not 3 beta-HSD type II (M(r), 44K) expressed in fetal testis. In cultures of fetal zone and definitive zone cell of human fetal adrenal, 3 beta-HSD expression was not detected until ACTH was added. ACTH, likely acting in a cyclic AMP-dependent process, induced 3 beta-HSD type II activity and mRNA expression. The higher level of 3 beta-HSD mRNA in definitive zone compared with fetal zone cells was associated with parallel increases in cortisol secretion relative to dehydroepiandrosterone sulfate formation.

3-Hydroxysteroid Dehydrogenases↗
PubMed1993

Multiple isoforms of 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase in mouse tissues: male-specific isoforms are expressed in the gonads and liver.

Multiple isoforms of 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase (3 beta HSD) are expressed in various mouse tissues in a tissue-specific, sex-specific, and developmental manner. Three distinct immunoreactive species [molecular masses, 47, 44, and 42 kilodaltons (kDa)] are detectable by Western immunoblot analysis using a 3 beta HSD antiserum. Different immunoreactive isoforms are expressed in steroidogenic (44 and 47 kDa in gonads) and nonsteroidogenic (42 and 47 kDa in liver and kidney) tissues. Two of these isoforms are sex-specific in the gonads (47 kDa) and liver (42 kDa), because they are detectable only in male mice. Sex-specific expression in the liver is developmentally regulated. Low levels of this male-specific hepatic isoform are first detectable at 23-25 days of age, but its level of expression increases progressively during sexual maturation to adult levels. NAD(+)-dependent 3 beta HSD activity is detectable in homogenates of all tissues examined, but the kinetic characteristics of this activity differ among tissues and are sexually dimorphic in the liver. Apparent Michaelis constants for dehydroepiandrosterone are much lower in steroidogenic (0.24 +/- 0.07 microM for testis) than in nonsteroidogenic (range, 10-100 microM for liver and kidney) tissues and are lower in male mouse liver (16 +/- 1 microM) than in female mouse liver (82 +/- 20 microM). Oligonucleotides with unique sequences but encoding homologous regions of the mouse type I, II, and III 3 beta HSD cDNAs were used for Northern blot analyses. A type I oligomer hybridizes with RNA from steroidogenic (adrenal, ovary, and testis) tissues, and a type III oligomer hybridizes with RNA from nonsteroidogenic (liver and kidney) tissues. A type II oligomer, however, hybridizes specifically with RNA from testis and liver of male mice, tissues that express a male-specific 3 beta HSD. These results suggest that type II-like transcripts may encode a 47-kDa sex-specific 3 beta HSD in testis and a 42-kDa sex-specific 3 beta HSD in liver of male mice. It is unclear how many members of subfamilies of the 3 beta HSD gene family will be discovered. The mouse may prove to be a valuable experimental model, as this is the first species in which multiple immunoreactive isoforms can be identified in a single tissue. This multiplicity makes it difficult to correlate the size and number of immunoreactive isoforms with the diverse kinetic characteristics of NAD(+)-dependent 3 beta HSD activities in tissue homogenates and to develop isoform-specific probes.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMed1991

3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase expression in rat and characterization of the testis isoform.

The isolation, cloning and expression of a DNA insert complementary to mRNA encoding rat testis 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase (3 beta-HSD) is reported. The insert contains an open reading frame encoding a protein of 373 amino acids, which exhibits 73% and 78% identity to the cDNA encoding the human placental form at the amino acid and nucleotide levels respectively. Northern blot analysis of total RNA of rat tissues using as probe a specific radiolabeled cDNA insert encoding rat testis 3 beta-HSD demonstrated high levels of 1.6 kb mRNA species in ovary, adrenal and Leydig tumor, with lower but detectable message in testis and adult male liver, while the probe also hybridized to a 2.1 kb mRNA species in liver. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to 3 beta-HSD present in H540 Leydig tumor cell homogenate and human placental microsomal 3 beta-HSD, as detected by immunoblot analysis, and catalyzed the conversion of pregnenolone to progesterone, 17 alpha-hydroxypregnenolone to 17 alpha-hydroxyprogesterone, and dehydroepiandrosterone to androstenedione. Transfected COS cell homogenates, supplemented with NAD+, but not NADP+, converted pregnenolone to progesterone and dehydroepiandrosterone to androstenedione with apparent Km values of 0.13 and 0.09 microM, respectively. Immunoblot analysis of various rat tissues using a polyclonal antibody directed against human placental 3 beta-HSD, in addition to immunoreactivity in the adrenal and testis, demonstrated immunoreactive 3 beta-HSD protein in adult male liver, but not in adult female or fetal liver. We conclude that while one gene product is highly expressed in testicular Leydig cells, and probably adrenal and ovary, accounting for their 3 beta-HSD content, a 3 beta-HSD is also expressed in liver in a sex-specific manner.

Amino Acid Sequence↗
PubMed1991

Corticotropin regulation of 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase in ovine adrenocortical cells: inhibition by transforming growth factor beta.

In the steroidogenic pathway, 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) is a key enzyme which controls the formation of delta 4-3-ketosteroids from delta 5-3-beta-hydroxysteroids. Herein, we used primary cultures of ovine adrenocortical (OAC) cells to study the effects of ACTH and transforming growth factor beta (TGF-beta) on 3 beta-HSD activity, protein and mRNA levels. TGF-beta has been previously reported to be a potent inhibitor of steroid formation in OAC cells. By using an antibody against human placental 3 beta-HSD, we showed that ACTH-treatment had a dose- and time-dependent stimulatory effect on 3 beta-HSD protein amount. This effect was maximal using 10(-9) M ACTH after a 48 h treatment. When included in the treatment medium, TFG-beta inhibited this stimulation by ACTH in a dose- and time-dependent manner. We also used a human 3 beta-HSD cDNA probe to demonstrate that the effect of both ACTH and TFG-beta were exerted at the mRNA level with maximal effects observed using 10(-9) M for ACTH and 1 ng/ml for TGF-beta. Bu2cAMP mimicked the effects of ACTH, and TGF-beta had an inhibitory effect on this stimulation. It appears from these data that TGF-beta is a negative regulator of 3 beta-HSD expression in OAC cells. The inhibitory effect of TGF-beta on 3 beta-HSD was contrasted to the TGF-beta effect on 17 alpha-hydroxylase cytochrome P-450 (P-45017 alpha). While the levels of both enzymes decreased, that of 3 beta-HSD was less sensitive than that of P-45017 alpha which decreased following TGF-beta treatment to non-detectable levels. The different sensitivities of steroidogenic enzymes to factors which regulate growth and differentiation such as TGF-beta may play a role in determining the nature of steroids released from adrenocortical cells.

Adrenal Glands↗
PubMed1991

Regulation of 3 beta-hydroxysteroid dehydrogenase in adrenocortical cells: effects of angiotensin-II and transforming growth factor beta.

The maintenance of optimal steroidogenesis in adrenocortical cells primarily depends on the chronic action of ACTH to promote the synthesis of the various steroid metabolizing enzymes. In the steroidogenic pathway, the ratio of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) to 17 alpha-hydroxylase cytochrome P450 (P-450(17 alpha)) plays a key role in determining the final steroid products released by adrenal cells. The differences in these enzymes are particularly important when one considers the adrenal zones and the secretion of the zone-specific steroids. In the present study we have investigated the regulation of 3 beta HSD with regard to its enzyme activity, levels of protein and changes in specific mRNA encoding for this enzyme. Following eight days in primary culture, bovine adrenocortical (BAC) cells were found to respond to both ACTH and Bu2 cAMP by increased cortisol production. In addition, 3 beta HSD activity, enzyme protein and mRNA levels were increased in response to both factors. The increases varied from 2-fold for activity to 5-7 fold for mRNA. ACTH and Bu2cAMP also greatly increased P-450(17 alpha) from the near undetectable levels in control cells. In order to examine the possibility of differential regulation of these adrenal steroidogenic enzymes we determined the effects of angiotensin II (A-II) and transforming growth factor beta (TGF beta) on the levels of these enzymes. Both of these factors decreased the ACTH-stimulated levels of P-450(17 alpha) enzyme and mRNA to near nondetectable levels observed within control cells. In addition, these compounds inhibited the ACTH induction of 3 beta HSD. While the mechanism of TGF beta action is not clear, A-II probably is acting through protein kinase C. Indeed the protein kinase C activating phorbol ester, TPA, mimicked the inhibitory effects of A-II on 3 beta HSD and P450(17 alpha). It is important to point out, however, that the effects of A-II and TGF beta on P450(17 alpha) activity appeared more pronounced than their action of 3 beta HSD. This observation may relate to the relative stability of 3 beta HSD as compared to P450(17 alpha). Taken together these data indicate that, while A-II and TGF beta each decrease the levels of steroid-metabolizing enzymes, a differential regulation is observed in that P-450(17 alpha) protein and activity levels are much more sensitive to treatment with these factors.

3-Hydroxysteroid Dehydrogenases↗
PubMed1991

Regulation of 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase expression by adrenocorticotropin in bovine adrenocortical cells.

In the steroidogenic pathway, 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta HSD) catalyzes the formation of hormonally active delta 4-3-ketosteroids from delta 5-3 beta-hydroxysteroids. In the present study the regulation of 3 beta HSD by ACTH action on bovine adrenocortical (BAC) cells in primary culture was evaluated. Western blot analysis was accomplished using an antibody against human placental 3 beta HSD. The relative molecular mass of 3 beta HSD in these cells was 45K, which was similar to that in human placenta. A significant effect of ACTH was not detected until day 6 of culture due to the high basal levels of the enzyme in BAC cells. Treatment of cells with ACTH on day 8 of culture resulted in a marked increase in the amount of 3 beta HSD protein, and this effect was correlated directly with enzymatic activity. The effects of ACTH were time and dose dependent, with an increase detectable only after 48 h of treatment; the maximal response was obtained with 10(-9) M ACTH. As demonstrated by Northern analysis, ACTH action was manifested by increasing the steady state level of 3 beta HSD mRNA. A human 3 beta HSD cDNA probe, which was used in this study, hybridized to a 1.7-kilobase species of BAC RNA. The effects of ACTH on 3 beta HSD activity and increases in 3 beta HSD protein and mRNA in BAC cells were mimicked by treatment with (Bu)2cAMP. The findings of this study suggest that ACTH controls 3 beta HSD gene expression in BAC cells by a cAMP-dependent mechanism similar to that involved in the expression of steroid hydroxylase genes. However, because the different stabilities of 3 beta HSD and hydroxylase proteins and/or mRNAs may play a critical role in determining the zone-specific steroids secreted from the adrenal cortex, other cAMP-dependent or independent regulatory mechanisms may also be important in regulating the expression of adrenal 3 beta HSD.

Adrenal Cortex↗
PubMed1991

Prostaglandin E2 is a positive regulator of adrenocorticotropin receptors, 3 beta-hydroxysteroid dehydrogenase, and 17 alpha-hydroxylase expression in bovine adrenocortical cells.

The maintenance of optimal steroidogenesis in adrenocortical cells primarily depends on the chronic action of cAMP. Herein we examine the effects of prostaglandin E2 (PGE2) on the differentiated functions of bovine adrenocortical (BAC) cells in primary culture. PGE2 (10 microM) treatment for 3 h stimulated steroidogenesis and cAMP production by over 100-fold. In addition, the cAMP antagonist Rp-cAMP (1 mM) inhibited PGE2 stimulation of steroidogenesis by 60%. This observation suggests that the cAMP second messenger system is responsible for much of the PGE2-activated steroid hormone synthesis. Chronic treatment of BAC cells with PGE2 caused induction of 3 beta-hydroxysteroid dehydrogenase and steroid 17 alpha-hydroxylase cytochrome P-450 expression as determined by the examination of enzyme activity, enzyme levels by immunoblotting, and specific messenger RNA (mRNA) levels by Northern analysis. The positive effects of PGE2 on expression of 3 beta-hydroxysteroid dehydrogenase and 17 alpha-hydroxylase cytochrome P-450 were similar to the effects seen after ACTH treatment of BAC cells. In addition, treatment of BAC cells with PGE2 for 3 days caused a 3-fold induction of ACTH receptors as determined by increased cell binding of [125I]ACTH. Finally, we determined that BAC cells produced PGE2 and that the level of synthesis increased 10-fold after treatment with the hormone angiotensin II. Taken together these data indicate that PGE2 is a positive regulator of BAC cell differentiation acting on ACTH receptors, steroid metabolizing enzymes, and steroidogenesis. The ability of BAC cells to produce PGE2 leaves open the possibility for paracrine and autocrine regulation within the adrenal.

3-Hydroxysteroid Dehydrogenases↗
PubMed1991

Regulation of expression of male-specific rat liver microsomal 3 beta-hydroxysteroid dehydrogenase.

In the steroidogenic pathways present in the gonads and adrenal cortex, 3 beta-hydroxysteroid dehydrogenase isomerase (3 beta HSD) is a key enzyme which controls the formation of delta 4-3-ketosteroids from delta 5-3 beta-hydroxysteroids. Herein, we used an antibody against human placental 3 beta HSD and a rat testicular 3 beta HSD cDNA probe to study the expression of rat liver 3 beta HSD mRNA and protein. Rat liver microsomal 3 beta HSD activity has been previously reported to exhibit a significant sex difference, with much higher activity in the male. We have shown an age-dependent increase in levels of immunoreactive 3 beta HSD through the time of maturation of the male rat. The immunoreactive protein, of similar molecular size to the human placental and rat testicular 3 beta HSD, was localized to the microsomal fraction of liver and was concentrated in pericentral locations. Immunoreactive protein was not detected in liver of immature (before 25 days of age) rats of either sex or in adult female liver. Northern blot analysis of liver and testicular RNA with a rat testicular 3 beta HSD cDNA probe revealed the presence of a 1.6-kilobase mRNA species in addition to the major 2.1-kilobase mRNA species in adult male liver, neither of which was detected in immature or adult female liver RNA. Hypophysectomy of female rats or treatment with testosterone implants caused induction of liver 3 beta HSD protein, while continuous infusion of GH to male rats decreased the level of 3 beta HSD protein. Similarly, the levels of the mRNA species were decreased after GH treatment. Using [3 alpha-3H]dehydroepiandrosterone as substrate for 3 beta HSD activity, we determined the apparent Km for liver microsomal NAD(+)-dependent 3 beta HSD activity to be 20 microM in both adult male and female liver and was much greater than the Km of rat Leydig tumor 3 beta HSD activity (0.2 microM). Liver 3 beta HSD activity was inhibited by trilostane, a proven inhibitor of gonadal and adrenal 3 beta HSD activity. A rat liver 3 beta HSD cDNA was isolated from a male liver cDNA library that was closely related to the type II 3 beta HSD form of rat ovary but different from type III liver 3 beta HSD. The enzyme obtained upon expression of this cDNA had properties characteristic of male-specific NAD(+)-dependent liver microsomal 3 beta HSD (i.e. high apparent Km for dehydroepiandrosterone) and distinct from those of the high affinity gonadal type I 3 beta HSD.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

Control of production of insulin-like growth factor I by pig Leydig and Sertoli cells cultured alone or together. Cell-cell interactions.

The production of insulin-like growth factor I (IGF-I) by pig Leydig cells and pig Sertoli cells cultured alone or together was investigated. Human chorionic gonadotropin (hCG) and basic fibroblast growth factor (FGF) stimulate in a dose-dependent manner IGF-I production by Leydig cells. At maximal concentrations the effects of both factors were almost additive. Insulin at micromolar concentrations enhanced IGF-I production and potentiated the effects of hCG and FGF. The secretion of IGF-I by Sertoli cells was stimulated by FSH and FGF. Under basal conditions, the production of IGF-I by the coculture was similar to the addition of the production by each cell cultured alone. In contrast, in the presence of hCG, FSH or FGF, the production of IGF-I by the coculture largely exceeded that expected from the monocultures. Moreover, stimulation of the coculture with both hCG and FSH resulted in a further increase in IGF-I production. These results indicate that Leydig as well as Sertoli cells secrete IGF-I and that the secretion of both cell types is stimulated by the corresponding gonadotropin. In addition, they indicate that Leydig-Sertoli interactions play a role in the control of IGF-I production, supporting the contention that this growth factor plays an important role in the paracrine and autocrine control of testicular functions.

Animals↗
PubMed1990

Transforming growth factor-beta inhibits steroid 17 alpha-hydroxylase cytochrome P-450 expression in ovine adrenocortical cells.

The maintenance of optimal steroidogenesis in adrenocortical cells primarily depends on the chronic action of ACTH to promote the synthesis of the various steroid-metabolizing cytochrome P-450 enzymes. In the steroidogenic pathway, 17 alpha-hydroxylase cytochrome P-450 (P-450(17) alpha) is a key enzyme controlling the formation of cortisol and androgens. Recently, we demonstrated that transforming growth factor-beta (TGF beta) is a potent inhibitor of steroid production in ovine adrenocortical cells. In the present study we used a polyclonal antibody to P450(17) alpha to determine adrenal cell P-450(17) alpha enzyme content by Western analysis. In addition, we used a cDNA probe encoding for bovine P-450(17) alpha mRNA to determine levels of P-450(17) alpha mRNA in sheep ovarian adrenocortical cells in primary culture. When cells were cultured in a serum-free medium in the presence of ACTH for 48 h, P-450(17) alpha activity, enzyme content, and mRNA levels for P-450(17) alpha increased by 3- to more than 10-fold. TGF beta decreased the basal level and completely blocked the stimulatory action of ACTH on P-450(17) alpha enzyme activity. The effects of TGF beta on P-450(17) alpha enzyme content and mRNA levels were manifested in a dose-dependent manner, with maximal inhibition observed using 1 ng/ml TGF beta. Importantly, the inhibitory effects of TGF beta on P-450(17) alpha were not overcome by (Bu)2cAMP. These findings indicate that TGF beta is a potent negative regulator of P-450, and the inhibitory action appears to be at the level of P-450(17) alpha gene expression. The ability of TGF beta to suppress the positive stimulatory action of ACTH suggests that TGF beta could play a role in determining the pathway of steroidogenesis and, thereby, the specific steroids secreted by adrenocortical cells.

17-alpha-Hydroxyprogesterone↗
PubMed1989

Hormonal regulation of insulin-like growth factor I secretion by bovine adrenal cells.

The role of insulin-like growth factor I (IGF-I) on the specific function of several steroidogenic cells has been recently reported. Since IGF-I is produced by several tissues, we have investigated whether bovine adrenal cells secrete this peptide. Purification of conditioned medium from adrenal cells incubated with [35S]methionine through affinity chromatography (monoclonal anti-IGF-I antibody), high pressure liquid chromatography, and polyacrylamide gel electrophoresis revealed a single band of similar Mr as pure recombinant IGF-I. Moreover, the purified adrenal-secreted IGF-I displaced bound 125I-IGF-I to its adrenal receptors, and pretreatment of adrenal cells with the purified peptide enhanced the acute corticotropin (ACTH)-induced cAMP production as recombinant IGF-I. The basal secretion of IGF-I (6 +/- 1 ng/48 h/10(6) cells) was stimulated 3-, 4.5-, and 9.5-fold by fibroblast growth factor, angiotensin II (A-II), and ACTH, respectively, but not by growth hormone. The stimulatory effects of A-II and ACTH were dose-dependent (ED50 congruent to 2.5 x 10(-8) and 1.5 x 10(-10) M, respectively), and the effects of both hormones were additive. Glucocorticoids were not the mediators of the effect of the two hormones on IGF-I secretion, since inhibition of their steroidogenic action by aminoglutethimide did not significantly modify IGF-I secretion. An immunoreactive IGF-I material was also secreted by mouse adrenal tumor cell line Y-1, but the stimulatory effect of ACTH was only 2-fold, and there was no effect of A-II. Since bovine adrenal cells contain specific IGF-I receptors and this peptide is required for the maintenance of some adrenal cell-specific function, the present data suggest that IGF-I may act in an autocrine fashion to stimulate adrenal cell differentiation stimulated by ACTH and A-II.

Adrenal Glands↗